Taxonomic identification and life cycle comparison of two populations of the monostromatic green algae Monostroma nitidum

Abstract Monostroma nitidum, a monostromatic green algae (MGA) with high economic value, is distributed worldwide. Life cycle often serves as a fundamental criterion for taxonomic classification. Most researchers consider the life cycle of M. nitidum to involve dimorphic alternation of generations, although the possibility of a monomorphic asexual life cycle remains unclear. In this study, tufA and 18S rDNA sequences were employed as molecular markers, complemented by morphological analysis, to classify and identify MGA in two distinct habitats: Hailing Island reefs (YJ) and Naozhou Island reefs (ZJ). The results of tufA and 18S rDNA sequence analysis revealed that all samples from YJ and ZJ clustered to the same branch (M. nitidum clade) with high bootstrap support and genetic distances of less than 0.000 and 0.005, respectively. However, morphological observations indicated significant differences in the external morphology of the YJ and ZJ samples, although both initially exhibited a filament‐blade form during early development. The life cycle of the ZJ samples exhibited typical dimorphic alternation of generations, whereas the YJ samples only produced biflagellate asexual gametes with negative phototaxis. Gametes of the YJ samples directly developed into new gametophytes without undergoing the sporophyte stage. Consequently, the YJ and ZJ samples were classified as monomorphic asexual and dimorphic sexual M. nitidum, respectively. These findings provide evidence supporting the monomorphic asexual life cycle of M. nitidum for the classification of MGA.


| INTRODUC TI ON
Marine macroalgae are photoautotrophic organisms without roots, stems, leaves, or embryos.To date, more than 140 kinds of edible marine macroalgae rich in minerals, vitamins, and dietary fiber have been described (Klnc et al., 2013;Pereira, 2011), Marine macroalgae are consumed as traditional foods in Asian countries (Mouritsen et al., 2019) and used as important sources of raw materials for production of pharmaceuticals, chemicals, and animal feed (Leandro et al., 2019).In addition, marine macroalgae has continued to attract increasing attention in aquaculture.In 2020, global production of cultured algae reached 35.1 million tons (wet weight), accounting for approximately 40% of the total aquaculture production worldwide of 87.5 million tons (FAO, 2022).The dominant species of cultured marine macroalgae are red seaweed (Rhodophyta) and brown seaweed (Heterokontophyta), while green seaweed accounts for less than 1% of global production.However, production is closely monitored for only eight genera of cultured marine macroalgae, which include Ulva, Enteromorpha, Caulerpa, and Monostroma (Cai et al., 2021).
Among these, Monostroma production is only monitored in South Korea with a total output of 6321 tons, accounting for 37% of the total production of green seaweed worldwide.Large-scale production of Monostroma began in Japan more than 60 years ago with annual output ranging from 1400 to 2500 tons of dry product in 1960 (Kida, 1990;Ohno, 1995;Ohno & Triet, 1997).However, Monostroma production has not been reported in the World Fisheries Yearbook for many years, possibly because of the close genetic relationships among numerous types of green seaweed, which can easily lead to difficulty in classification (Lewis & McCourt, 2004).For instance, green seaweed of the genera Ulva and Monostroma are registered in the Food and Agriculture Organization database as "bright green nori" or "green laver," thus reflecting a certain degree of taxonomic uncertainty (Moreira et al., 2022).In addition, the diversity in external morphology, early development, and reproductive modes has intensified challenges associated with systematic classification and commercial categorization.
Members of the genus Monostroma (Chlorophyta, Ulvophyceae) stand out as the most cultivated species of green seaweed with widespread distribution in warm inner bays and estuaries (Bast et al., 2009a(Bast et al., , 2009b;;Leliaert et al., 2012), and are valued for delicious taste and rich nutritional value (Gupta et al., 2015;Nisizawa et al., 1987;Pise et al., 2012).Monostroma polysaccharide extract is widely used in the production of marine medicines, healthcare products, cosmetics, and various industrial applications (Hoang et al., 2015;Karnjanapratum & You, 2011;Lee et al., 2010;Tako, 2017).Japan and South Korea boast the largest cultivation of Monostroma, while commercialization endeavors have also commenced in Brazil (Pellizzari & Reis, 2011), India (Kavale et al., 2020), and select regions of China (Chen et al., 2019).Nevertheless, there is no consensus regarding the classification of Monostroma.
In 1854, Thuret classified all monostromatic green algae (MGA) into the genus Monostroma (Ulvaceae) (Thuret, 1854), thus many MGA species were subsequently assigned to this genus.In 1934, Kunieda separated the genus Monostroma from the family Ulvaceae based on the life cycle of heterotypic alternation of generations and established the family Monostromaceae (Kunieda, 1934).Since Thuret did not select a type species when establishing Monostroma, Monostromaceae was based on the typical life cycle characteristics of M. latissimum (Bast, 2015).In view of the polymorphism and life cycle of MGA, scholars have persistently debated the attributes of MGA, leading to prolonged confusion in classification.Based on the presence of intermediate forms of filament, disc, and sac structures during early development of the thallus, as well as considerations of life cycle types, sexual or asexual reproductive modes, classification of MGA has undergone numerous revisions and divisions, resulting in the formation of seven genera (Kaur et al., 2023;Papenfuss, 1960;Tatewaki, 1969): Monostroma, Ulvaria, Ulvopsis, Kornmannia, Protomonostroma, Gayralia, and Capsosiphon (Table 1).(Pellizzari et al., 2013).In fact, macroalgae commonly exhibit both sexual and asexual reproduction (Hiraoka, Dan, et al., 2003).For example, some species of the genus Ulva do not undergo normal meiosis or the combination of germ cells, but rather directly complete the life cycle through asexual reproduction (Hiraoka, Shimada, et al., 2003).Species with an asexual life cycle are believed to have lost the ability to undergo sexual differentiation for a second time during evolution (Van Den Hoek et al., 1995), as environmental coercion has been identified as the driving force behind the change in reproductive strategy of Ulva species (Ichihara et al., 2019).However, the evolutionary trajectory of sexual and asexual reproduction of MGA species remains unclear.

Advancements in molecular biology
An earlier study by our group analyzed the external morphological features and ITSs sequences of MGA from various locations along the coast of Guangdong, China (Cui et al., 2022).The results of the phylogenetic analysis revealed that MGA could be categorized into three branches: Monostroma nitidum, Gayralia brasiliensis, and Monostroma sp.The genetic distance between the genera Monostroma and Gayralia is smaller than the internal genetic distance within the genus Monostroma with primary differences in frond thickness and size.However, multigene marker identification and life cycle studies have not been conducted previously.In genome and evolutionary research pertaining to large green algae, the tufA gene and 18S rDNA stand out as prominent | 3 of 16 LIAO et al. molecular marker genes.Studies have shown that the tufA gene, notable for its absence of introns, boasts a high success rate in amplification and demonstrates proficient species identification capabilities, it is deemed suitable as a DNA barcode marker for conducting phylogenetic analyses of large green algae (Saunders & Kucera, 2010).Additionally, 18S rDNA serves as a valuable tool for elucidating the genetic relationships among large green algae and contributes significantly to the establishment of an accurate classification system.It serves as a pivotal data source for inferring the phylogenetic relationships within the realm of green algae (Leliaert et al., 2012).Therefore, the foundations of early research in the laboratory were combined with external morphological and life cycle characteristics, along with tufA and 18S rDNA genetic markers of two populations of MGA collected from different habitats to provide theoretical support to elucidate potential evolutionary relationships for the classification of MGA.

| Collection of MGA samples
MGA samples were collected from wild reefs located on Naozhou Island (ZJ; 20°56′33″ N, 110°36′32″ E) and Hailing Island (YJ; 21°38′41″ N, 111°53′53″ E) on March 8 and 11, 2023, respectively (Figure 1).The two sampling sites are both located at the edge of a mangrove.There are no buildings around the ZJ habitat and the substrate is mainly composed of reefs and gravel.The water is highly transparent with a temperature of 24°C, salinity of 32‰, and pH of 8.10.The YJ habitat is located on the edge of a rock dam where a creek enters the sea.The bottom material is mainly sediment and the water is turbid with a temperature of 28.0°C, salinity of 30‰, and pH of 7.84.
Sediments and impurities were removed from the surface of the MGA samples using sterilized seawater and brushes.

| Extraction and sequencing of tufA and 18S rDNA
Total DNA was extracted from three randomly selected YJ samples (YJ-1, YJ-2, and YJ-3) and ZJ samples (ZJ-1, ZJ-2, and ZJ-3) using the Ezup Column Super Plant Genomic DNA Kit (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China) in accordance with the manufacturer's instructions.Two genes (tufA and 18S rRNA) were amplified and sequenced (Lin et al., 2013;Saunders & Kucera, 2010).The universal primer sequences and cited references are detailed in Table 2.Each reaction volume for polymerase chain reaction (PCR) analysis included 1 μL each of the forward and reverse primers (10 μmol/L), 1 μL of DNA template, 1 μL of dNTP Mix (10 mmol/L), 2.5 μL of 10× Taq buffer, and 0.2 μL of Taq DNA polymerase (5 U/μL) with ddH2O added to a total volume of 25 μL.The reaction conditions included a predenaturation step at 95°C for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 63°C for 30 s, and extension at 72°C for 30 s. Subsequently, repair and extension were conducted at 72°C for 10 min, which was followed by a final incubation step at 4°C. (NJ) and maximum-likelihood (ML) methods (Saitou & Nei, 1987) with 1000 bootstrap replicates to assess branch confidence (Felsenstein, 1985).Evolutionary distances were calculated using the maximum composite likelihood model (Tamura et al., 2004) and the units are the number of base substitutions per site.The conserved motifs of the tufA and 18S rDNA gene sequences from the samples were predicted using the online motif prediction tool MEME (https:// meme-suite.org/ meme/ ), with the number of motifs searched set to 20.

| Morphological observation and cell culture
External morphological observations of 30 randomly selected MGA samples each from YJ and ZJ were conducted.The length, width, and color of the MGA samples were recorded.The MGA samples were manually sectioned with a razor.Micrographs of gametocyte cross sections and surface cells were captured using a microscope (BX53; Olympus Corporation, Tokyo, Japan).Gametocyte thickness and vegetative cell size were measured.To obtain gametes, the YJ and ZJ samples were cultured separately (temperature, 25°C; light intensity, 60 μmol photons m −2 s −1 ; photoperiod, 12-h light: dark) to induce gametophyte maturation.
After about 1 week, visually identifiable gametophyte edge cells transitioned from yellow-green to yellow-brown.Microscopic observation revealed the formation of numerous gametocysts.
Gametophytes with yellow-brown edges were collected and dried in the shade at room temperature for 12 h.Then, the gametocytes were stimulated with unilateral light (100 μmol photons m −2 s −1 ) to release gametes.Utilizing the phototaxis characteristics of gametes, To obtain zoospores from the ZJ samples, mature sporangia were maintained in the dark for 2 weeks and then stimulated with unilateral light (100 μmol photons m −2 s −1 ) to release zoospores.Leveraging the phototaxis characteristics of zoospores, purified zoospore liquid was collected, diluted, and observed under a microscope to identify zoospores morphologically while recording the details.The zoospores were cultivated consistent with the method for culturing gametes or zygotes mentioned above.

| Statistical analysis
All data are presented as the mean ± standard deviation.Statistical  3 and 4).Bast (2015).The genetic distance between the ZJ and YJ samples was equal to the intraspecific genetic distance of Ulva fasciata (DQ286547, AB425964) (0.002), which was smaller than the interspecific genetic distance between M. grevillei and M. angicava (0.000-0.004) (Tables 3 and 4).Furthermore, the motifs of the tufA and 18S rDNA gene sequences were further analyzed through the MME online website, revealing that the tufA and 18S rDNA gene sequences are highly conserved, with their distribution and positions on the sequence being essentially consistent (Figures 3 and 4).
TA B L E 3 Genetic distances based on the tufA sequences of the YJ and ZJ samples with the reference sequences retrieved from the GenBank.

| Comparative analysis of morphological features
There were extremely significant differences in morphological features between the YJ and ZJ samples (p < .01)(Table 5).The YJ gametophytes were characterized by yellow-green coloration and smaller dimensions with an average length of 9.21 ± 1.84 cm and width of 7.97 ± 2.23 cm.In contrast, the ZJ gametophytes were light green in color and larger in size with an average length of 36.18 ± 12.22 cm and width of 17.88 ± 5.17 cm (Figures 5a and 6a).Microscopically, the vegetative cells of the YJ and ZJ samples were predominantly arranged in pairs, with distinctly visible nuclei (Figures 5b and   6b).The YJ vegetative cells were round with an average length of 7.11 ± 0.88 μm and width of 5.57 ± 0.81 μm.In contrast, the ZJ vegetative cells were oval with longer dimensions, featuring an average length of 8.37 ± 1.10 μm and width of 4.81 ± 0.85 μm.Notably, the average cross-section thickness of the YJ samples was approximately half that of the ZJ samples (16.26 ± 1.08 vs. 32.40 ± 2.07 μm, respectively; Figures 5c and 6c).
The ZJ gametes were dioecious, while YJ gametes were asexual.
Nonetheless, both were collectively released through dissolution and shedding of mature gametocysts at the edges of the gametophytes.There was no trace of the cell wall remaining after the release of the gametes by the gametocysts.Statistically, there were significant differences between the YJ and ZJ gametes (p < .01) in both length (6.04 ± 0.51 vs. 4.10 ± 0.50 μm, respectively) and width (2.42 ± 0.32 vs. 2.10 ± 0.32 μm, respectively).The YJ and ZJ gametes had two anterior flagella and exhibited irregular movements after release (Figures 5d and 6d).However, the ZJ gametes moved more rapidly and for longer periods than the YJ gametes.The ZJ gametes exhibited strong positive phototaxis after release but were switched to negative phototaxis after the male and female gametes had combined.Following cessation of movement, the flagella separated and formed a ball.In contrast, the YJ gametes exhibited weak positive phototaxis after release and rapidly shifted to negative phototaxis approximately 5 min later with no observed binding of gametes from different samples.

| Life cycle studies
The life cycle of the YJ samples was monomorphic and asexual (Figure 7b).Gametes appeared as single spherical cells about 3 μm in diameter after 12 h of fixation (Figure 5e).The nuclei began to divide at 24 h (Figure 5f) and formed two cells after 48 h (Figure 5g).
However, the process of binary fission was often uneven.The nucleus first moved to one end and divided horizontally upward to form daughter cells, while the mother cell grew downward to form F I G U R E 3 Motif analysis of tufA sequences of YJ and ZJ algal strain samples.
the initial rhizome.At approximately day 5, the gametes developed into uniseriate filament germlings (Figure 5h).On day 17, longitudinal division occurred.Cells in the middle of the uniseriate germlings began to spread toward both ends and a filamentous rhizome became observable at the base (Figure 5i).As longitudinal division progressed, the gametes differentiated into multiple rhizoids by day 24 (Figure 5j) and formed a monolayer of germlings by day 36 (Figure 5k).Ultimately, germlings with diameters of 0.4 mm formed by about day 60 (Figure 5l).Notably, no saccate structures were observed during the early development of gametes.The ontogenetic form of the progeny remained consistent with that of the parents.
The life cycle of the ZJ samples followed the typical sexual dimorphic alternation of generations with the ability to undergo parthenogenesis (Figure 7a).The gametes or zygotes were spherical after fixation (Figure 6f) and underwent slow development and exhibited binary fission.Approximately 2 month later, the gametes or zygotes formed sporangia with cell walls.After fixation for 180 days, the sporangia appeared as round or oval cells with clearly visible cell walls and diameters of approximately 25 μm (Figure 6g).Following 2 weeks of cultivation in the dark, the color of the sporangia transitioned from green to yellow-green and inner granular zoospores were observed (Figure 6h).In response to intense light stimulation, the zoospores broke through the convex wall of the sporangia and were collectively released.The released sporangia appeared as empty shells (Figure 6i).
The zoospores (mean length, 5.14 ± 0.71 μm; mean width, 3.42 ± 0.57 μm) exhibited strong positive phototaxis and had four superior flagella (Figure 6j).During the early developmental stage, the ZJ zoospores morphologically resembled the YJ gametes with a filament-blade ontogenetic form but exhibited faster growth.After 12 h of fixation, the zoospores formed spherical cells with diameters of 4 μm (Figure 6k).Unequal division occurred after 24 h, resulting in the formation of two cells (Figure 6l) and uniseriate germlings were observed at 48 h (Figure 6m).Longitudinal division occurred  on day 7 (Figure 6n).Around day 24, the zoospores formed a monolayer of germlings (Figure 6o) and eventually matured into germlings with diameters of 2.5 mm by day 36 (Figure 6p).The life cycle of the ZJ offspring and parents was the same.

| Recommendations for classification of MGA
The early classification of five genera of MGA (Ulvaria, Ulvopsis, Kornmannia, Protomonostroma, and Capsosiphon) was reasonably based on morphological and life cycle characteristics.Analyses of the tufA and 18S rDNA sequences in this study indicate that the aforementioned five genera formed branches independent of M. nitidum (Figure 2a,b), in agreement with the phylogenetic analysis of MGA reported in 2015 by Bast (2015).The genetic distances among these branches based on the tufA and 18S rDNA sequences were 0.181-0.283and 0.014-0.099,respectively (Tables 4 and 5).The genus Monostroma established by Thuret in 1854 originally referred to MGA with frond lengths of 2-30 cm, although there was no designated model species.M. bullosum, characterized by a heterotypic alternation of generations, and M. oxyspermum, displaying monomorphic asexuality, are defined as the original species of the genus Monostroma (Bast, 2015;Thuret, 1854).Later studies proposed various classifications of MGA.For example, in the mid-1960s, Gayral (1964Gayral ( , 1965) ) advocated for the classification of heteromorphic alternating generations species (M.grevillei) within the genus Monostroma, which fol- as Pseudothrix groenlandica based on the results of a conjoint analysis of ribosomal SSU and ITS sequences (Hanic & Lindstrom, 2008).
There is no obvious taxonomic difference between the asexual M. nitidum and the G. brasiliensis, although the latter exhibits positive phototaxis after the release of gametes.The only difference between the two and G. oxysperma is the absence of saccate structures in the early developmental stage, the life cycle of G. oxysperma is consistent with the parthenogenetic life cycle of Monostroma wittrockii.In addition, there are currently only three species in the genus Gayralia, including G. kuroshiense cultivated in Korea, which was added by Andiska et al. (2023).In fact, G. kuroshiense was originally named M. kuroshiense, which refers to the collective name of M. latissimum and M. nitidum (Bast, 2015).
Based on the results of morphological and molecular analyses,  5), and the life histories exhibit marked distinctions (Figures 5 and 6), as M. nitidum of the YJ population only exhibits asexual reproduction.Intriguingly, despite these pronounced differences in morphological and life cycle characteristics, there was no apparent genetic distinction between the two populations, as determined by phylogenetic analysis (Figure 2a,b).The influence of environmental factors on macroalgae morphology (Carrington, 1990;Littler & Littler, 1980) and reproductive strategies (Fierst et al., 2010;Searles, 1980) has been previously reported.Under low-salinity conditions, MGA exhibit earlier maturation as compared to high-salinity conditions (Kida, 1990).MGA in well-sheltered habitats are longer than those in areas with faster currents and larger waves (Bast et al., 2009a).Additionally, sterile conditions can cause abnormal early development (Matsuo et al., 2003).
According to a report by Tatewaki (1969) and a study published by O'kelly and Floyd (1984), MGA have 14 life cycle types and none seem to exhibit a single characteristic (e.g., filament, saccate, or discoid intermediate) for differentiation from other genera, which reflects the high degree of phenotypic plasticity of MGA, at least to some extent.The environmental conditions of the YJ habitat seem to be more severe than those of the ZJ habitat.The YJ habitat is located on the edge of the rock dam at an estuary of a creek, thus salinity significantly fluctuates, and the substrate primarily consists of mud and gravel, resulting in relatively high turbidity.This environment is similar to the "geographic parthenogenesis" pattern described by Haag and Verduijn (Haag & Ebert, 2004;Verduijn et al., 2004).In marginal habitats, populations frequently undergo cycles of local extinction and subsequent recolonization.Genetic bottlenecks commonly arise during the recolonization phase, culminating in the production of asexual propagules.Asexual species of many plants and animals are more common in marginal habitats, such as high latitudes or altitudes, where the potential for adaptive evolution is greater (Fagerström & Poore, 2001).Previous reports have documented instances of asexual M. latissimum in regions characterized by significant salinity fluctuations (Bast et al., 2009b).M. latissimum was previously thought to only exist as a sexual species.Furthermore, the asexual biflagellate zoids produced by Ulva prolifera demonstrate a preference for lowsalinity environments (Hiraoka & Higa, 2016).These zoids simultaneously exhibit two mating types genomes and are thought to have evolved through apomeiosis, a process that does not involve chromosome reduction in the sporophyte (Ichihara et al., 2019).
In this study, environmental stress was more severe in the YJ habitat, thus the existence of asexual biflagellate zoids may have resulted from adaptation to more severe environmental fluctuations.Asexual M. nitidum may also have evolved in the YJ habitat via apomeiosis.Consequently, asexual M. nitidum might be a secondary characteristic that evolved from sexual ancestors.Given a suitable environment, there is potential for asexual M. nitidum to re-evolve into a sexual species.Future research at the genetic level will be instrumental in gaining a deeper understanding of the relationship between reproductive strategies and the evolution of MGA species.

| CON CLUS ION
This study employed molecular markers and morphological studies of two populations of M. nitidum collected from YJ and ZJ.The results revealed no obvious differences in tufA and 18S rDNA sequences between the two populations, although there were extremely significant have introduced new techniques for identification and classification of MGA species, including sequencing of the internal transcribed spacer (ITSs) region, tufA, rbcL, 18S rRNA genes, and randomly amplified polymorphic DNA.In 2009, Bast et al. reported that the early development of M. latissimum was characterized by a filament-blade asexual life cycle (Bast et al., 2009a, 2009b).Analysis of nuclear-encoded rDNA revealed consistency in the ITS1 sequences of asexual and sexual M. latissimum.Therefore, Bast et al. suggested that it was unreasonable to base the genera Protomonostroma and Gayralia on the typical asexual life cycle, and proposed that members of the genus Gayralia should be reclassified to the genus Monostroma.In 2013, Pellizzari et al. advocated to classify M. latissimum into the genus Gayralia along with other species of the genus Monostroma Locations of MGA sample collection and the state of YJ and ZJ samples in the natural habitats.
purified gamete fluid was collected, diluted, and examined for gamete morphology under a microscope.The sex of the gametes was determined by observation of the fusion of gametes from different MGA samples.Finally, the gametes or zygotes were inoculated into a culture dish and subjected to a 12-h dark: light photoperiod at a constant temperature of 20°C for even deposition and fixation to the dish bottom.Then, the gametes or zygotes were incubated at a constant temperature of 20°C, light intensity of 50 μmol photons m −2 s −1 , and 12-h light: dark photoperiod.Sterilized seawater was replaced every 3 days.Photographic images were captured regularly to record development.
analyses (one-way analysis of variance and Duncan's multiple comparison test) were performed using IBM SPSS Statistics for Windows, version 25.0.(IBM Corporation, Armonk, NY, USA).A probability (p) value <.05 was considered statistically significant.Graphs were generated using MEGA 11 software (https:// www.megas oftwa re.net/ ) and Origin 2021 software (https:// www.origi nlab.com/ 2021).3| RE SULTS3.1 | Homology analysis based on tufA and 18S rDNA sequencesAll YJ and ZJ samples were identified as M. nitidum, as there were no obvious genetic differences.The tufA and 18S rDNA sequences of G. brasiliensis and M. nitidum were homologous.Specifically, the genetic distance between the 18S rRNA gene samples of YJ and ZJ (Y-18S 1-3, Z-18S 1-3) is less than 0.0001, and the distance between all such samples and the reference sequence of M. nitidum (AF499665) is merely 0.002.For the tufA gene samples, the YJ samples (Y-tufA 1-3) exhibit a genetic distance of less than 0.0001 when compared with the reference sequences of Gayralia sp.(JF680967) and G. brasiliensis (MW242795, OR597303).Similarly, ZJ's tufA samples (Z-tufA 1-3) demonstrate a genetic distance of less than 0.0001 with reference sequences of M. nitidum (NC072924) and G. brasiliensis (NC072923).The genetic distance between the tufA samples of YJ and ZJ (Y-tufA 1-3, Z-tufA 1-3) is only 0.005.Additionally, genetic distances between the M. nitidum clade and the genera Kornmannia, Ulvopsis, and Protomonstroma range, respectively, between 0.070 ~ 0.074, 0.202 ~ 0.204, and 0.274 ~ 0.279 (Tables A phylogenetic tree of the samples was constructed based on the tufA and 18S rDNA sequences (Figure 2a,b), which included six genera of MGA classified as the Monostromaceae clade and members of the genus Ulvaria were classified as the Ulvaceae clade.Within the Monostromaceae clade, the species of Protomonostroma, Capsosiphon, and Kornmannia formed three independent branches that were strongly supported by bootstrap values.Two species classified to the genus Ulvopsis (i.e., M. angicava and M. grevillei) formed an independent branch of the 18S tree and a large branch of the tufA tree.All YJ and ZJ samples were clustered in the M. nitidum clade for both the tufA and the 18S maps.Bootstrap support values were all 100% and the genetic distance between the two was 0.005 and 0.000, respectively.Analysis of the tufA sequences showed that the YJ samples shared the same sequences as two species of the genus Gayralia, G. brasiliensis (OR597303, MW242795) and Gayralia sp.(JF680967), and the sequences of ZJ samples were the same as those of M. nitidum and G. brasiliensis.The genetic distance between the YJ and ZJ samples was equal to the intraspecific genetic distance of Ulvaria obscura (MH308689, MZ892916) and Ulva lactuca (MN322753, HQ610366) (both, 0.005).The model species of Gayralia, G. oxysperma (HQ610252), shared the same sequence as the Kornmannia model species.Additionally, the sequences of M. angicava (MG646366) and M. harriotii (MK507438, MK507443) were identical.However, M. harriotii may have been misidentified here.The 18S rDNA sequences of all ZJ and YJ samples were identical to those of Gayralia sp.(JF680952, JF680951) and maintained a genetic distance of 0.002 with M. nitidum (AF499665) and M. kuroshiense (GU062568, GU062566) within the genus Monostroma (M.kuroshiense being the collective name for M. nitidum and M. latissimum, as designated by

F
I G U R E 2 Phylogenetic tree derived from the tufA (a) and 18S rDNA (b) sequences of the YJ and ZJ samples, along with sequences obtained from GenBank, is presented.Experimental samples are highlighted in bold.Bootstrap values from NJ (left) and ML (right) analyses are displayed on the branches.Only bootstrap values exceeding 50% are noted.

F
Motif analysis of 18S rDNA sequences of YJ and ZJ algal strain samples.TA B L E 5 Morphological characteristics of the YJ and ZY samples.
lows a disc-sac-blade development pattern in the early life cycle, into the genus Ulvopsis.Simultaneously, the monomorphic alternation of generations species (M.obscurum) with a filament-sac-blade pattern is classified in the genus Ulvaria.The genus Monostroma would then consist of only the asexual species M. oxyspermum, characterized by somatic cells arranged in pairs or fours and the collective release of gametes through the rupture of gametocysts.Kornmann (1964) argued that the early development of disc-sac-blade is characteristic of the genus Monostroma and the asexually reproduced M. oxyspermum, which develops into a filament-sac-blade in the early stage, F I G U R E 5 Morphology and ontogeny of the YJ thallus.(a-c), Gametophyte morphology: (a) Gametophyte; (b) Vegetative cells; (c) Gametophyte cross section.(d) Gametes at 1 h after release.(e-l) Germination of gametes: (e) 12 h after release.(f) 24 h after release.(g) 48 h after release.(h) 5 days after release.(i) 17 days after release.(j) 24 days after release.(k) 36 days after release.(l) 60 days after release.should be excluded from the genus Monostroma.From a morphological and anatomical point of view, in 1968, Bliding suggested that M. zostericola and M. leptodermum, which have microscopic discoid intermediates and leafy sporophytes, should be classified into the new genus Kornmannia (Bliding, 1968), although the latter only produces quadrifiagellate asexual zoids.In 1969, Vinogradova classified M. groenlandicum into the genus Capsosiphon and established two new genera, Protomonostroma and Gayralia (Vinogradova, 1969), and classified the asexually reproduced species M. undulatum and M. oxysperma as the model species Protomonostroma undulatum and G. oxysperma, respectively.However, C. groenlandicus has been classified

F I G U R E 6
phototaxis.The early developmental stage was consistent with sexual we suggest that MGA should be classified into six genera: namely, Ulvaria, Ulvopsis, Kornmannia, Protomonostroma, Capsosiphon, and Monostroma.Following the naming priority principle of the International Code of Nomenclature for algae, fungi, and plants(Turland et al., 2018), G. oxysperma and G. kuroshiense should retain the original names of M. oxyspermum and M. kuroshiense, while G. brasiliensis should be renamed M. nitidum, as all three should be classified to the genus Monostroma.4.2 | Thoughts on the morphological differences of MGA caused by two habitatsPhenotypic plasticity and genetic variation are important factors in the adaptive evolution of MGA.The emergence of asexual M. nitidum is the result of environmental selection, they are secondary and have lost the capacity for sexual reproduction for the second time over a long evolutionary period.In this study, significant disparities were observed in the external morphological characteristics of M. nitidum between the YJ and ZJ populations (Table
Genetic distances based on the 18S rDNA sequences of the YJ and ZJ samples with the reference sequences retrieved from the GenBank.
TA B L E 4